Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt

ABSTRACT

The present invention relates to a composition useful for the diagnosis of diseases associated with aberrant expression of the genes encoding the secreted proteins Futrin 1, 2, 3 and/or 4(=R-Spondin 2, 3, 1 and 4, respectively), e.g. in connection with tumors or diseases of the muscle, kidneys or bones. The present invention also relates to a pharmaceutical composition containing a compound which is capable of modifying (a) the expression of the gene encoding Futrin 1, 2, 3 and/or 4 or (b) the activity of the Futrin 1, 2, 3 and/or 4 protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/951,152, filed Jul. 25, 2013 which is a divisional of U.S.application Ser. No. 13/931,481, filed Jun. 28, 2013, which is adivisional of U.S. application Ser. No. 10/575,217, filed Apr. 10, 2006,the contents of both of which are hereby incorporated by reference,which is a national stage application of International Application No.PCT/EP2004/011269 having an international filing date of Oct. 8, 2004and which claims benefit under 35 U.S.C. §119 to European PatentApplication No. 0302300.7 filed Oct. 10, 2003.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 3, 2014, isnamed P5660D3C1_Sequence_Listing.txt and is 31,571 bytes in size.

BACKGROUND

The present invention relates to compositions useful for the diagnosisand therapy of diseases associated with aberrant expression of the genesencoding the proteins Futrin 1, 2, 3 and/or 4 (=R-Spondin 2, 3, 1 and 4,respectively). These diseases include tumors of e.g. the breast, ovary,liver, uterus, cervix, colon, lung, ovary, rectum, testis, pancreas,bones and skin, as well as diseases involving muscle, bone, lipid andglucose metabolism, and obesity. The present invention also relates to apharmaceutical composition containing a compound which is capable ofmodifying (a) the expression of the gene encoding Futrin 1, 2, 3 and/or4 or (b) the activity of Futrin 1, 2, 3 and/or 4.

The Wnt signal cascade plays a crucial role as regards regulation ofsurvival, proliferation and differentiation of cells duringembryogenesis, and in the adult as shown, e.g., in Drosophila, Xenopusand mice (Nusse and Varmus, Cell 69 (1992), 1073-1087). Wnt-genes encodesecretory glycoproteins which activate a well characterized signalcascade via a Wnt receptor called “frizzled”.

The Wnt signalling cascade and its components also play an importantrole in various diseases which makes it desirable to modulate itsactivity:

i) Cancer

Tumorigenesis represents a complex multistage process in which geneticchanges and environmental factors are thought to deregulate the cellularprocesses that control cell proliferation and differentiation. Severalstudies indicate that an aberrant Wnt signal cascade is involved in thedevelopment of colon cancer, breast cancer and melanoma (Pfeifer,Science, 2 75 (1997), 1752-1753; Polakis, Genes Dev. 14 (2000),1837-1851). The first gene encoding a protein of the Wnt signal cascade,int-1, was isolated from mouse mammary tumor virus (MMTV) and it couldbe shown that it is an oncogene. It is thus well established that anaberrant regulation of the activity of Wnt and/or components of the Wntsignal cascade downstream of the Wnt signal, e.g., beta-catenin and APC,are involved in tumorigenesis.ii) Bone DiseaseWnt signals promote bone formation (e.g. Yang, Development, 130(2003),1003-15; Fischer, J. Biol. Chem. 277 (2002) 30870-30878). Consistentwith this notion, a gain-of-function mutation of the Wnt receptor LRP5causes high bone disease (Boyden, et al., 346 (2002) N Engl J Med,1513-21; Little, et al., 70 (2002) Am J Hum Genet, 11-9). Conversely,inactivating mutations in LRP5 leads to osteoporosis-pseudogliomasyndrome in humans (Kato, et al., 157 (2002) J Cell Biol, 303-14; Gong,et al., 107 (2001) Cell, 513-23).iii) Eye DiseaseInactivating mutation in the Wnt receptor LRP5 lead to pseudoglioma inhumans and eye malformations in mice (Kato, et al., 157 (2002) J CellBiol 303-314; Gong, et al., 107 (2001) Cell, 513-523).iv) KidneyAberrant Wnt signalling is involved in renal fibrosis (Surendran, Am JPhysiol Renal Physiol 282 (2002) 431-441) and polycystic kidney disease(Saadi-Kheddouci, Oncogene 20 (2001) 5972-5981).v) Lipid and Glucose Metabolism, ObesityDeficiency of the Wnt receptor LRP5 in mice leads to increased plasmacholesterol levels in mice fed a high-fat diet, because of the decreasedhepatic clearance of chylomicron remnants. In addition, when fed anormal diet, LRP5-deficient mice show a markedly impaired glucosetolerance (Fujino, et al., 100 (2003) Proc Natl Acad Sci USA, 229-234.)Administration of the LRP5 antagonist Dkk1 to mice reduces glucoseuptake in various cell line and decreases fat deposition (WO 02/066509).

It is thus clear from the above that the Wnt signalling pathway isinvolved in a variety of human diseases. Yet, means for the therapy ordiagnosis of diseases associated with a dis-regulated Wnt signal cascadeare insufficiently available. Thus, the use of reliable diagnosticmolecular markers would be helpful for an understanding of the molecularbasis of diseases associated with an aberrant Wnt signal cascade. It canbe expected that such markers are also useful for therapy and for thedevelopment of novel therapeutic avenues for treatment of Wnt signalcascade dependent diseases, as detailed above.

Thus, the technical problem underlying the present invention is toprovide means for diagnosis and therapy of diseases associated with anaberrant Wnt signal cascade.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

During the experiments resulting in the present invention four genes,futrin 1, 2, 3 and 4, could be identified the products of which aremodulators of the Wnt pathway. Futrin 2 was previously identified ashPWTSR (Chen et al., 29 (2002), Mol. Biol. Rep. 287-292), a protein ofbefore unknown role or function, expressed in numerous cell types.Further, human Futrin 1, 2, 3, and 4 were described as Stem Cell GrowthFactor-Like Polypeptides, which are able to promote proliferation ofhematopoietic stem cells (WO-A-01/77169; WO-A-01/07611).

SUMMARY

In the present invention the following is shown for the first time: 1)Futrins enhance Wnt signalling and this is of physiological relevancesince inhibition of Futrin 1 or 2 results in inhibition of the Wntsignal cascade (Wnt/(β-catenin signalling). These data show that Futrinscan be regarded as Wnt modulators. Futrin 1 (Rspo2) is coexpressed withand positively regulated by Wnt signals and synergizes with Wnt toactivate β-catenin. Analysis of functional interaction with componentsof the Wnt/β-catenin pathway suggests that Rspo2 functionsextracellularly at the level of receptor ligand interaction. AntisenseMorpholino experiments in Xenopus embryos and RNAi experiments in HeLacells revealed that Rspo2 is required for Wnt/β-catenin signalling. InXenopus embryos depleted of Rspo2 the muscle markers myoD and myf5 failto be activated and later muscle development is impaired. The resultsindicate that Rspo2 is a novel activator of the Wnt/β-catenin cascade.Thus, Futrins like Rspo2 (Futrin 1) are useful for the diagnosis and thedevelopment of therapies for Wnt-LRP mediated diseases, including butnot limited to tumor suppression, bone formation, cholesterol andglucose metabolism (including diabetes), obesity, kidney disease and eyedisease. 2.) Since the data obtained show that Futrin 1 is required formuscle formation Futrin 1 is useful for the diagnosis and thedevelopment of therapies for muscle related diseases, including muscleregeneration. 3.) The data show that Futrins are aberrantly expressed ina variety of human tumors. Thus, Futrins are useful for tumor diagnosisand the development of cancer therapies. For example it has been foundout that in most of the tumors the expression of Futrins 1-3 isdramatically decreased (colon, stomach, lung, rectum tumors for Futrin1, breast, ovary, bladder, uterus, cervix, rectum tumors for Futrin 2,uterus and cervix tumors for Futrin 3). In a few cases the expression ofFutrin 1-3 is upregulated (one case of stomach tumor for Futrin 1 and 2,ovary tumor for Futrin 3). Futrin 4 shows very low level of expressionin most of the tissues studied, except ovary.

Thus, the inhibition of the Wnt signal cascade by inhibiting theexpression/activity of Futrins or by stimulating the expression/activityof the Futrins will have a therapeutic effect. Likewise, the activationof the Wnt signal cascade by decreasing the expression of Futrin and/orby repressing the activity of the polypeptide itself will have atherapeutic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Identification of Xenopus Futrin 1 (Rspo2). TOP-FLASH reporter,vector DNA from pools of 250 clones each and pCSfrizzled8 wereco-transfected. Luciferase reporter assays in 293T cells were carriedout in 96 well plates in duplicates as described (Wu et al., Curr Biol10 (2000), 1611-1614). Luciferase activity was normalized againstcotransfected Renilla-luc activity using a commercial kit (Clonetech).RLU: relative luciferase units.

FIG. 2A-F: Futrin 1 (Rspo2) promotes Wnt/β-catenin signaling. (FIG. 2A)Identification of Xenopus Rspo2 by expression screening. An expressionscreen was carried out in 293T cells transfected with a Wnt-responsivereporter (TOPFLASH) and plasmid DNA pools from a Xenopus eye cDNAlibrary. RLU: relative light units. (FIG. 2B) The C-terminus of Rspo2mediates cell surface retention. Myc-tagged Rspo2 (wt) or Rspo2ΔC (ΔC)were transfected in 293T cells, and cell lysate and medium were analysedby Western blot. Co, untransfected cells. (FIG. 2C) Rspo2 activatesWnt/β-catenin signalling. TOPFLASH luciferase reporter assays werecarried out in 293T cells with the following transfected DNAs: mouseWnt1 (Wnt), 5 ng; mouse frizzled8(fz), 1 ng; Xenopus Rspo2 (Rspo2), 0.1,0.3, 0.9 and 2.7 ng. (FIG. 2D) Rspo2 enhances Wnt3a signalling. MouseWnt3a, Xenopus Rspo2ΔC (Rspo2ΔC) or mock-conditioned media were added to293T cells followed by TOPFLASH luciferase reporter assays. (FIG. 2E)Rspo2 stabilizes β-catenin. (top) 293T cells were treated with Rspo2ΔC,Wnt3a or mock-conditioned media for 1 or 4 hours as indicated. Cytosolicfractions were subjected to Western blot and probed for β-catenin andα-tubulin (loading control). (bottom) immunohistochemical staining ofβ-catenin in SHEP cells after 3 hour treatment with the conditionedmedia indicated. Arrowheads indicate nuclear β-catenin. The percentageof cells showing the represented staining is 90% (Co), 85% (Rspo2), 80%(Wnt3a) and 90% (Wnt3a+Rspo2). (FIG. 2F) Domain analysis of Rspo2. (top)schematic drawing of Xenopus Rspo2 and deletion constructs, sp, signalpeptide; FU1, 2, furin-like domains; TSP1, thrombospondin type 1 domain;C, positively charged C-terminus. (bottom). TOPFLASH luciferase reporterassays were carried out in 293T cells with the indieated constructs.Equal protein production was confirmed by Western blot (data not shown).

FIG. 3A-D: Multisequence nucleic acid alignment of cDNAs encoding humanFutrin 1 (SEQ ID NO:21) Futrin 2 (SEQ ID NO:33), Futrin 3 (SEQ ID NO:20)and Futrin 4 (SEQ ID NO:23) and Xenopus Futrin 1 (SEQ ID NO:24).Identical nucleotides are highlighted in grey. All nucleic acidsequences begin with the translation initiator ATG codon indicated withan asterisk.

FIG. 4: Multisequence amino acid alignment of human Futrin 1 (SEQ IDNO:26), Futrin 2 (SEQ ID NO:27), Futrin 3 (SEQ ID NO:34) and Futrin 4(SEQ ID NO:28) deduced from human cDNAs (see FIG. 3). Identical aminoacids are highlighted in grey, similar amino acids are in dotted box.

FIG. 5A-B: Futrins promote Wnt signaling. Cotransfection experiments in293T cells. (FIG. 5A) Wnt-responsive luciferase reporter assays wereperformed in 96 well plates in triplicates as described (Wu et al., CurrBiol 10 (2000), 1611-1614). Luciferase activity was normalized againstRenilla activity using a commercial kit (Clonetech). (FIG. 5B)Wnt1=mouse Wnt1, fz8=mouse frizzled8, Futrin1=xenopus Futrin 1,Wnt3A=mouse Wnt3A, (0.1 ng) in A indicates amount of plasmid DNAtransfected per well, RLU: relative luciferase units.

FIG. 6A-C: Sequence comparison of human Rspo proteins (hR-spondin 1 to4). (FIG. 6A) Alignment of human (h) Rspo proteins (SEQ ID NOS:29-32,respectively, in order of appearance) (corresponding to the alignmentsof human Fut1-4 in FIG. 4 except that different designations are used).The signal peptide, furin-like domains and thrombospondin type 1 domainare underlined and conserved amino acids are shown in grey. (FIG. 6B,FIG. 6C) Rspo homology matrix showing overview of amino acid identity in% between human Rspo proteins (FIG. 6B) and between Xenopus (X) or mouse(m) Rspo2 and human Rspo proteins, respectively (FIG. 6C).

FIG. 7: Human Futrin 1 and 2 are required for Wnt signaling. Hela cellswere transfected in 24-well plates with the Wnt reporter7LEF-Rev-fosLuc, pRL-TK and pSuper plasmids that produce either siRNAagainst human Futrin1 and 2 or a nonsense control. Three days aftertransfection, mouse Wnt3A conditioned medium was added to the culture tostimulate Wnt signalling. 24 hours later, luciferase activity wasdetermined.

FIG. 8A-M: Expression analysis of Xenopus and mouse R-spondins, (FIG.8A) Rspo2 expression during Xenopus development at the indieatedembryonic stages analysed by RT-PCR. Histone H4 was used fornormalization. −RT, minus reverse transcription control. (FIG. 8B-H)Xenopus whole mount in situ hybridizations of the indicated genes. (FIG.8B) Stage 11 embryo, dorso-vegetal view; dbl, dorsal blastoporal lip.(FIG. 8C) Stage 12 embryo, dorsal view with anterior up. An anteriorneural expression domain is indicated by arrowhead. (FIG. 8D) Stage 15embryo, dorsal view with anterior up. (FIG. 8E) Stage 14 embryo, dorsalview with anterior up. (FIG. 8F-G) Tailbud stage embryos; ba, branchialarches; cm, cranial musculature; di, diencephalon; dnt, dorsal neuraltube; mhb, midbrain-hindbrain boundary; ov, otic vesicle; pn,pronephros; pdm, proctodeum; s, somites; tb, tailbud mesoder. Inset in(F) shows a transverse section at the level indicated by arrowhead,showing expression in dorsal neural tube and in the dorsal- andventral-most parts of the somites. (FIG. 8H) Dissected Xenopus tadpolebrain (lateral view) showing expression in diencephalons (di) and zonalimitans intrathala ica (zli), where sonic hedgehog is expressed(inset). dt, vt, dorsal and ventral thalamus, respectively, sc, spinalcord; tel, telencephalon; (FIG. 8I-M) Mouse whole mount in situhybridizations of the indicated genes. (FIG. 8I) limb buds of day 12.5mouse embryos. AER, apical ectodermal ridge. (FIG. 8J) Day 7.5 mouseembryo showing Rspo3 expression in the primitive streak. (FIG. 8K-M) Day9.5 mouse embryos. di, diencephalon; dnt, dorsal neural tube; inet,metencephalon; tel, telencephalon.

FIG. 9A-E: Regulation of Xenopus R-spondins by Wnt signaling. (FIG. 9A)Comparison of XRspo2, XWnt8 and XWnt3a expression pattern in earlyneurula embryos by whole mount in situ hybridization. Dorsal view,anterior up. (FIG. 9B) (top) Diagram of experiment. Four cell stageembryos were injected with 50 pg pCS-ppl (preprolactin), pCSXWnt8 orpCS-β-catenin into each blastomere, DMZs were dissected, cultured untilstage 11 equivalent and analyzed by RT-PCR. (Bottom) RT-PCR analysis ofthe indicated genes. −RT, minus reverse transcription control. (FIG.9C-E) Four cell stage embryos were injected with 50 pg pCS-Wnt8,pCS-Wizt3a or pCS-ppl into one blastomere and fixed at stage 11 for insitu hybridisation with XRspo2.

FIG. 10A-C: Futrin 2 is required for muscle formation. FIG. 10AA-FIG.10AL. Depletion of Futrin 1 protein causes downregulation of earlymuscle markers and muscle defects. (FIG. 10AA, FIG. 10AC, FIG. 10AE,FIG. 10AG, FIG. 10Al, FIG. 10AK) Embryos injected with controlmorpholino oligo (5 ng), with Fut1-Mo (5 ng). Oligos were mixed withlineage tracer mRNA (LacZ) (blue staining) in (FIG. 10AC, FIG. 10AD,FIG. 10AE, FIG. 10AF, FIG. 10AG, FIG. 10AH) or preprolactin (ppl) RNA,visualised by in situ hybridisation (red staining) (FIG. 10AA, FIG.10AB, FIG. 10AJ, FIG. 10AK, FIG. 10AL). In situ hybridization withprobes to Xmyf5 (FIG. 10AA, FIG. 10AB), XmyoD (FIG. 10AC, FIG. 10AD),Xbra (FIG. 10AE, FIG. 10AF), Xnot (FIG. 10AG, FIG. 10AH) and muscleactin (FIG. 10AI-L) (magenta staining). Embryos in FIG. 10AK and FIG.10AL are cut transversally. Note reduction of muscle in FIG. 10AL (rightside). FIG. 10B. Fut1-Mo act specifically to inhibit translation oftheir cognate DNA constructs when overexpressed in embryos. mRNA(C-terminal myc tagged fut1) was injected equatorially in bothblastomeres at 2-cell stage embryos. The same embryos were then injectedat the 8-cell stage with 5 ng of Fut1-Mo (lane3) or control morpholino(lane2) in all vegetal blastomeres and harvested at stage 11. TaggedFutrinb protein was then visualised with a-myc antibody. FIG. 10C.Rescue of muscle marker reduction caused by Futl-Mo by coinjection ofXFut1 mRNA containing point mutations in the 5′region corresponding tomorpholino oligo. Embryos were coinjected with 5 ng of Futl-Mo (1, 2)with 50 pg of ppl or XFut1 mRNA radially in one blastomere at 4-cellstage.

Expression of MyoD (1, 3) and Myf5 (2, 4) were analysed by in situhybridization. All embryos were grouped into 3 classes (examples forMyoD are shown on the bottom of the figure): embryos with expressionlevel on the injected side from 1-30% (class A); 30-70% (class B), and70-100% (class C) from normal level. Bars represent the percentage ofthe embryos corresponding to type A, B or C. (n for Fut-Mo+ppl: 57embryos for MyoD, 45 embryos for Myf5; n for Fut-Mo+mRNA Ffut1: 39embryos for MyoD and 41 embryo for Myf5).

FIG. 11A-N: Rspo2 promotes neural and muscle differentiation in Xenopus.(FIG. 11A) Four-cell stage embryos were injected animally with 100 pgRspo2, 100 pg Xwnt8, or 200 pg β-catenin mRNA in each blastomere. Atstage 8, animal caps were dissected, cultured until stage 18 equivalentand analyzed for expression of the indicated marker genes, −RT, minusreverse transcription control. (FIG. 11B) Two-cell stage embryos wereinjected with 100 pg Rspo2 or preprolactin (ppl) mRNA in eachblastomere. Note ectopic cement glands (arrowhead) and shortened bodyaxis in Rspo2 injected embryo. (FIG. 11C-J) Whole mount in situhybridisations of the indicated genes. Eight-cell stage embryos wereinjected with 100 pg of Rspo2 or ppl RNAs as indicated into one animalblastomere. LacZ mRNA was coinjected as lineage tracer in all panelsexcept FIG. 11C, FIG. 11G and FIG. 11H. (FIG. 11C-H) Stage 15 neurulaein anterior view. (FIG. 11I-J) Stage 11 gastrulae in vegetal view,dorsal up. (FIG. 11K-N) Rspo2 promotes muscle formation. (FIG. 11K)Diagram of the experiments. Four-cell stage embryos were injected with50 pg plasmid DNA constructs in all blastomeres, the indicated fragmentswere explanted at stage 10.5, cultured, and processed for whole mount insitu hybridisation (FIG. 11M) of RT-PCR (FIG. 11N). (FIG. 11L) Stage 40equivalent VMZ of LMZ explants. Note tail-like structures in VMZs fromRspo2 injected embryos. (FIG. 11M) In situ hybridization of stage 25VMZs for muscle actin. (FIG. 11N) RT-PCR analysis for the indicatedgenes in stage 25 equivalent VMZ and stage 11 equivalent DMZ explants.Xdd I, dominant negative. Xenopus dishevelled; Co, preprolactin.

FIG. 12A-D: Rspo2 interferes with BMP-4, Activin and Nodal but not withFGF in Xenopus. (FIG. 12A-D) Four-cell stage embryos were injectedanimally with indicated RNAs. At stage 8, animal caps were dissected,cultured until stage 10 equivalent and analyzed for expression of Vent2(FIG. 12A) or Xbra (FIG. 12B-D). −RT, minus reverse transcriptioncontrol. Embryos injected with 100 pg preprolactin (ppl) were used ascontrol. Amounts of mRNAs used: 100 pg Rspo2, 50 pg or 250 pg of BMP-4,50 pg of activin, 25 or 100 pg of Wnt8, 200 pg of β-catenin, 50 or 100pg of Xnrl, 2 or 20 pg of FGF8.

FIG. 13A-D: Xenopus Rspo2 is required for muscle formation. (FIG. 13A)Rspo2Mo specifically inhibits translation of its cognate DNA. Top,Diagram of the experiment. Two-cell stage Xenopus embryos were injectedwith 100 pg Myc-tagged Rspo2 mRNA in the animal region, at 8-cell stage,the same embryos were then injected with 5 ng of Rspo2Mo of CoMo in allanimal blastomeres, harvested at stage 11 and processed for Western blotanalysis of Myc-tagged Rspo2 and α-tubulin. (FIG. 13Ba-l) Depletion ofRspo2 protein causes muscle defects and down-regulation of myogeneicmarkers. Four-cell stage embryos were injected equatorially into oneblastomere with 5 ng control morpholino oligonucleotides (CoMo) orRspo2Mo as indicated together with 50 pg ppl mRNA or 50 pg LacZ RNA aslineage tracer and analysed at tailbud of gastrula stage by in situhybridization for the indicated genes. In (FIG. 13Ba-f) double in situhybridization for gene of interest (dark blue) and for ppl (red) wasused, (FIG. 13Ba-d) Stage 25 embryos. (FIG. 13Ba-b): myotomes,visualised by muscle actin expression show malformations an Rspo2Moinjected side (FIG. 13Bb). (FIG. 13Bc-d) Transversal section at thetrunk level showing reduced muscle volume in (FIG. 13Bd). (FIG. 13Be-h):myf5 and myoD expression (dark blue) is down-regulated in the Rspo2Moinjected region (red in FIG. 13Be, FIG. 13Bf of light blue in FIG. 13Bg,FIG. 13Bh). (FIG. 13Bi-l): Xbra and Xnot2 expression (dark blue) is notaffected in the region of Rspo2Mo injections (light blue). (FIG. 13C)Rspo2Mo acts specifically. Rescue of myf5 reduction by co-injected Rspo2mRNA. Four cell stage embryos were injected in one blastomere with 5 ngof control morpholino (CoMo), Rspo2Mo, 50 pg ppl mRNA of 50 pg Rspo2mRNA containing mismatches to Rspo2Mo. Expression of myf5 was analysedby in situ hybridization at gastrula stage. The percentage of embryoswith, strongly affected (A), moderately reduced (B) and normal myf5expression (C) as displayed in the representative embryos is indicated.Standard deviation was calculated from three independent experiments.(FIG. 13Da-d) Rspo2Mo Blocks Wnt signalling upstream of dishevel ledduring muscle formation. Four-cell stage embryos were radially injectedwith 5 ng of Rspo2Mo or CoMo, 50 pg pCS-XWnt8, pCS-dishevel led (Xdsh),pCS-dominant negative GSK-3β (dnGSK) or pCS-β-catenin. At stage 10.5DMZs (FIG. 13Da) or VMZs (FIG. 13Db) or LMZs (FIG. 13Dc-d) wereexplanted and cultured until stage 11 (FIG. 13Da, FIG. 13Dc) or 25 (FIG.13Db, FIG. 13Dd) for RT-PCR analysis of the genes indicated. −RT: minusreverse transcription control.

FIG. 14A-D: R-spondins are required for Wnt signalling in HeLa cells.(FIG. 14A) RT-PCR analysis showing differential expression of humanRspo1-4 in HeLa and 293T cell lines. Actin was used for normalisation.(FIG. 14B) Specificity of siRNAs. 293T cells were cotransfected withpSUPER-Rspo2 or -3 (siRNA), FLAG-tagged Rspo3 and GFP (transfectioncontrol). Expression of Rspo3 and GFP were analyzed by Western blot.(FIG. 14C-D) R-spondins are required for Wnt/β-catenin signalling inHeLa cells. Wnt luciferase reporter assay in HeLa cells cotransfectedwith the indicated constructs. Wnt3a was added as conditioned medium.RLU, relative light units.

FIG. 15A-E: Futrin expression is deregulated in various human tumors.(FIG. 15A-E) The expression of Futrin 1, 2, 3 and -4 or ubiquitin (toshow equal loading) was analysed by radioactive hybridisation on arrayedmRNAs (Clontech, Cancer Profiling Array II) from normal and canceroustissue samples from different patients. Abbreviations N, normal tissues;T, tumor tissues.

FIG. 16: Expression of human R-spondins in tumor samples. Dot blotanalysis of human Rspo1, 2 and 3 in normal and tumour samples. The sameCDNA samples (Cancer Profiling Array II, Clontech) were hybridized withhuman Rspo1, 2, 3 and ubiquitin probes, (top) The Cancer Profiling Arraycontains pairs of cDNAs from tumor and corresponding normal tissuesamples from individual patients and spotted side-by-side as indicated.Organ abbreviations are: Br, breast; ov, ovary; co, colon; st, stomach;lu, lungs; kidney; bi, bleeder, vu, vulva; pr, prostate; tr, trachea;liv, liver; ut, uterus; ce, cervix; re, rectum; th, thyroid gland; te,testis; sk, skin; sin, small intestine, pa, pancreas.

FIG. 17A-C: Interaction of R-spondins with components of theWnt/β-catenin signalling pathway. (FIG. 17A-C) Wnt luciferase reporterassay in 293T cells co-transfected with the indicated constructs.Results are indicated as fold-stimulation over reporter alone. DNA doseswere 5 ng Wnt1, 1 ng Fz8, 3 ng LRP6, 10 ng axin, 10 ng dntcf3, 5 ngdkk1, 10 ng Xdsh, 1 ng Wnt3a.

DETAILED DESCRIPTION

The present invention relates to a diagnostic composition comprising:

-   -   (a) at least one nucleic acid molecule comprising the nucleotide        sequence encoding Futrin 1, 2, 3 or 4 as depicted in FIG. 3;        and/or    -   (b) at least one polypeptide molecule comprising the amino acid        sequence encoding Futrin 1, 2, 3 or 4 as depicted in FIG. 4 or 6        a; and/or    -   (c) at least one nucleic acid molecule the complementary strand        of which hybridizes to a nucleic acid molecule of (a) and which        encodes a polypeptide with the biological activity of Futrin 1,        2, 3 or 4; and/or    -   (d) at least one fragment of (a), (b) or (c) having the        biological activity of Futrin 1, 2, 3 or 4;    -   (e) at least one nucleic acid molecule the sequence of which        differs from the sequence of the nucleic acid molecule of        (a), (c) or (d) due to the degeneracy of the genetic code,        and/or    -   (f) at least one ligand capable of specifically binding to the        molecule of (a), (b), (c), (d) or (e).

As used herein the term “polypeptide” not only refers to polypeptidesencoded by the nucleotide/amino acid sequences as depicted in FIGS. 3and/or 4 but also to polypeptides differing in amino acid sequence dueto insertion, deletion and/or substitution of one ore more amino acidsand showing at least one biological activity of a Futrin, e.g. theability to promote Wnt signalling. Preferably, the related nucleic acidsand/or polypeptides are nucleic acids and/or polypeptides the sequenceof which shows an identity of at least 40%, in particular an identity ofat least 65%, preferably of at least 80% and, particularly preferred, ofat least 90% to the amino acid sequences of the polypeptides encoded bythe nucleotide sequences shown in FIG. 3.

The nucleic acid molecules useful as probes can be both DNA and RNAmolecules, preferably they are single-stranded DNA molecules. They canbe isolated from natural sources or can be synthesized according to knowmethods.

As a hybridization probe nucleic acid molecules can be used, forexample, that have a nucleotide sequence which is exactly or basicallycomplementary to a nucleotide sequence as depicted in FIGS. 3 and 4 or 6a, respectively, or parts of these sequences. The fragments used ashybridization probe can be synthetic fragments that were produced bymeans of conventional synthetic methods.

As used herein, the term “hybridizing” relates to hybridization underconventional hybridization conditions, preferably under stringentconditions as described, for example, in Sambrook et al., MolecularCloning, A Laboratory Manual 2nd edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. However, in certain cases, ahybridizing nucleic acid molecule can also be detected at lowerstringency hybridization conditions. Changes in the stringency ofhybridization and signal detection are primarily accomplished throughthe manipulation of formamide concentration (lower percentages offormamide result in lowered stringency, salt conditions, or temperature.For example, lower stringency conditions include an overnight incubationat 37° C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 9.2MNaH₂PO₄; 0.02M EDTA, pH7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmonsperm blocking DNA, following by washes at 50° C. with 1×SSPE, 0.1% SDS.In addition, to achieve even lower stringency, washes performedfollowing stringent hybridization can be done at higher saltconcentrations (e.g. 5×SSC). Variations in the above conditions may beaccomplished through the inclusion and/or substitution of alternateblocking reagents used to suppress background in hybridizationexperiments. The inclusion of specific blocking reagents may requiremodification of the hybridization conditions described above, due toproblems with compatibility.

The term “ligand” as used herein refers to any molecule which is capableof specifically binding to Futrin 1, 2, 3, or 4, thus allowing todetermine the level of receptor molecules. Examples of such moleculesinclude antibodies, oligonucleotides, proteins or small molecules. Themolecule can be the natural ligand of Futrins, or can be closely relatedto said ligand, e.g., a fragment of the ligand, or a natural substrate,a structural or functional mimetic; see, e.g., Coligan, CurrentProtocols in Immunology 1(2) (1991); Chapter 5. In either case, themolecule can be isolated or rationally designed using known techniques;see also infra.

Preferably, the ligand is an antibody. The term “antibody”, preferably,relates to antibodies which consist essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations. Monoclonal antibodies are made from anantigen containing Futrin 1, 2, 3, or 4 or fragments thereof by methodswell known to those skilled in the art (see, e.g., Köhler et al., Nature256 (1975), 495). As used herein, the term “antibody” (Ab) or“monoclonal antibody” (Mab) is meant to include intact molecules as wellas antibody fragments (such as, for example, Fab and F(ab′) 2 fragments)which are capable of specifically binding to Futrin 1, 2, 3 and/or 4.Fab and f(ab′)2 fragments lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody. (Wahl et al., J. Nucl. Med. 24: 316-325(1983)). Thus, these fragments are preferred, as well as the products ofa FAB or other immunoglobulin expression library. Moreover, antibodiesof the present invention include chimerical, single chain, and humanizedantibodies.

For certain purposes, e.g. diagnostic methods, the nucleic acid moleculeused as probe or the ligand, e.g., antibody, can be detectably labeled,for example, with a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, oran enzyme.

The nucleic acid molecules can be used, for example, as probes orprimers in the diagnostic assays described below and allow, e.g., theanalysis of the expression of Futrin 1, 2, 3, or -4 by determining themRNA level or the determination of mutations within the coding region orregulatory regions leading to polypeptide molecules with altered, e.g.destroyed, activity, or leading to altered expression. Preferably, thenucleic acid molecules are oligonucleotides having a length of at least10, in particular of at least 15 and particularly preferred of at least50 nucleotides. These nucleic acid molecules of the invention can alsobe used, for example, as primers for a PCR reaction.

The present invention also relates to the use of a nucleic acid moleculeor ligand as defined above for the preparation of a diagnosticcomposition for the diagnosis of a disease associated with (a) aberrantexpression of Futrin 1, 2, 3, or -4 and/or (b) aberrant activity of aFutrin 1, 2, 3, or -4 polypeptide.

In a preferred embodiment, the target to which the nucleic acid moleculehybridizes is an mRNA.

The present invention also provides a method of diagnosing a diseaseassociated with (a) aberrant expression of Futrin 1, 2, 3, or -4 and/or(b) aberrant activities or amounts of a Futrin 1, 2, 3, or -4polypeptide in a subject comprising:

-   -   (a) determining (a) the amount of expression of Futrin 1, 2, 3,        or -4 and/or (b) the amount of biologically active Futrin 1, 2,        3 and/or 4 polypeptide in a biological sample; and    -   (b) diagnosing a disease associated with (a) aberrant expression        of Futrin 1, 2, 3 and/or 4 and/or (b) aberrant activities or        amounts of a Futrin 1, 2, 3 and/or 4 polypeptide or a risk for        the development of such disease based on an altered amount of        expression of Futrin 1, 2, 3 and/or 4 and/or (b) altered        activities or amounts of biologically active Futrin 1, 2, 3        and/or 4 polypeptide compared to a control.

Suitable assay formats are well known to the person skilled in the artand, in addition, described below. Suitable positive control samplesexpressing human Futrin proteins are, e.g., HEK 293 cells.

The Futrin 1, 2, 3, or 4 polypeptide or the corresponding mRNA, e.g. inbiological fluids or tissues, may be detected directly in situ, e.g. byin situ hybridization or it may be isolated from other cell componentsby common methods known to those skilled in the art before contactingwith a probe. Detection methods include Northern Blot analysis, RNaseprotection, in situ methods, e.g. in situ hybridization, in vitroamplification methods (PCR, LCR, QRNA replicase orRNA-transcription/amplification (TAS, 3SR), reverse dot blot disclosedin EP-B1 O 237 362), immunoassays, Western Blot and other detectionassays that are known to those skilled in the art.

The probe (e.g. a specific antibody or specific oligonucleotide) of thediagnostic composition can be detectably labeled. In a preferredembodiment, said diagnostic composition contains an anti-Futrin 1, 2, 3and/or 4 antibody and allows said diagnosis, e.g., by ELISA and containsthe antibody bound to a solid support, for example, a polystyrenemicrotiter dish or nitrocellulose paper, using techniques known in theart. Alternatively, said diagnostic compositions are based on a RIA andcontain said antibody marked with a radioactive isotope. Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium rhodamine, and biotin. In addition to assaying Futrin levelsin a biological sample, the polypeptide can also be detected in vivo byimaging. Antibody labels or markers for in vivo imaging of proteininclude those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma. A protein-specific antibody or antibody fragment which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ⁹⁹mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ⁹⁹mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific Futrin polypeptide. In vivo tumor imaging is, e.g.,described in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments”. (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

In a further aspect, the present invention, relates to a method foridentifying a binding partner to a Futrin 1, 2, 3 and/or -4 polypeptidecomprising:

-   -   (a) contacting-said polypeptide with a compound to be screened;        and    -   (b) determining whether the compound effects an activity of the        polypeptide.

The invention also includes a method of identifying compounds which bindto a Futrin 1, 2, 3 and/or 4 polypeptide comprising the steps of:

-   -   (a) incubating a candidate binding compound with said        polypeptide; and    -   (b) determining if binding has occurred.

Futrin 1, 2, 3 or -4 polypeptides may be used to screen for proteins orother compounds that bind to Futrin 1, 2, 3 or -4 or for proteins orother compounds to which Futrin1, 2, 3 and/or 4 bind. The binding ofFutrin1, 2, 3 or -4 and the molecule may activate (agonist), increase,inhibit (antagonist), or decrease activity of Futrin 1, 2, 3 or 4 or themolecule bound. Examples of such molecules include antibodies,oligonucleotides, proteins (e.g., ligands), or small molecules.

Preferably, the molecule is closely related to the natural ligand ofFutrin 1, 2, 3 or -4, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic; see, e.g.,Coligan, Current Protocols in Immunology 1(2) (1991); Chapter 5.

Preferably, the screening for these molecules involves producingappropriate cells which express Futrin 1, 2, 3 and/or 4 either as asecreted protein or on the cell membrane. Preferred cells include cellsfrom mammals, yeast, Drosophila, or E. coli. Cells expressing Futrin 1,2, 3 and/or -4 (or cell membrane containing the expressed polypeptide)are then preferably contacted with a test compound potentiallycontaining the molecule to observe binding, stimulation, or inhibitionof activity of Futrin 1, 2, 3 and/or -4.

The assay may simply test binding of a candidate compound to Futrin 1,2, 3 and/or 4, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to Futrin 1, 2, 3 and/or 4. Suitable assays to analyze theactivity of Futrin 1, 2, 3 and/or 4 include Wnt-inducible luciferasereporter assays in transfected HEK 293 cells, where Futrin 1, 2, 3and/or 4 synergizes with Wnt to enhance a Wnt-induced signal, such as isshown in FIG. 4.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining Futrin 1, 2, 3 and/or 4, measuring Futrin/molecule activityor binding, and comparing the Futrin/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure Futrin 1, 2, 3 and/or 4 level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure Futrin 1, 2, 3 and/or 4level or activity by either binding, directly or indirectly, to Futrin1, 2, 3 and/or 4 or by competing with Futrin 1, 2, 3 and/or 4 for asubstrate. All of these above assays can be used as diagnostic orprognostic markers. The molecules discovered using these assays can beused to treat disease or to bring about a particular result in a patient(e.g., elimination of a tumor, support of regenerative processes etc.)by modulating, preferably activating the Futrin 1, 2, 3 and/or 4molecule. Moreover, the assays can discover agents which may inhibit orenhance the production of Futrin 1, 2, 3 and/or 4 from suitablymanipulated cells or tissues.

Moreover, the invention includes a method of identifyingactivators/agonists or inhibitors/antagonists of a Futrin 1, 2, 3 and/or4 polypeptide comprising the steps of:

-   -   (a) incubating a candidate compound with said polypeptide;    -   (b) assaying a biological activity, and    -   (c) determining if a biological activity of said polypeptide has        been altered.

Suitable assays to analyze the activity of Futrin 1, 2, 3 and/or 4include Wnt-inducible luciferase reporter assays in transfected HEK 293cells, where Futrin 1, 2, 3 and/or 4 synergizes with Wnt to enhance aWnt-induced signal, such as is shown in FIG. 4.

In a further embodiment, the present invention relates to method ofidentifying and obtaining a drug candidate for therapy of diseasesassociated with (a) aberrant expression of Futrin 1, 2, 3 and/or 4and/or (b) aberrant activities or amounts of a Futrin 1, 2, 3 and/or 4polypeptide comprising the steps of

-   -   (a) contacting a Futrin 1, 2, 3 and/or 4 polypeptide or a cell        expressing said polypeptide, and optionally the corresponding        ligand (s), in the presence of components capable of providing a        detectable signal in response to binding to said drug candidate        to be screened; and    -   (b) detecting presence or absence of a signal or increase of the        signal generated, wherein the presence or increase of the signal        is indicative for a putative drug.

Suitable assays to analyze the activity of Futrin 1, 2, 3 and/or 4include Wnt-inducible luciferase reporter assays in transfected HEK 293cells, where Futrin 1, 2, 3 and/or 4 synergizes with Wnt to enhance aWnt-induced signal, such as is shown in FIG. 4.

The drug candidate may be a single compound or a plurality of compounds.The term “plurality of compounds” in a method of the invention is to beunderstood as a plurality of substances which may or may not beidentical.

Said compound or plurality of compounds may be chemically synthesized ormicrobiologically produced and/or comprised in, for example, samples,e.g., cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating Futrin1, 2, 3 and/or 4polypeptides. The reaction mixture may be a cell free extract or maycomprise a cell or tissue culture. Suitable set ups for the method ofthe invention are known to the person skilled in the art and are, forexample, generally described in Alberts et al., Molecular Biology of theCell, third edition (1994) and in the appended examples. The pluralityof compounds may be, e.g., added to the reaction mixture, culturemedium, injected into a cell or otherwise applied to a transgenicanimal. The cell or tissue that may be employed in the method of theinvention preferably is a host cell, mammalian cell or non-humantransgenic animal.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating a Futrin 1, 2, 3and/or 4 polypeptide, or one can further subdivide the original sample,for example, if it consists of a plurality of different compounds, so asto reduce the number of different substances per sample and repeat themethod with the subdivisions of the original sample. Depending on thecomplexity of the samples, the steps described above can be performedseveral times, preferably until the sample identified according to themethod of the invention only comprises a limited number of or only onesubstance(s). Preferably said sample comprises substances of similarchemical and/or physical properties, and most preferably said substancesare identical.

Several methods are known to the person skilled in the art for producingand screening large libraries to identify compounds having specificaffinity for a target. These methods include the phage-display method inwhich randomized peptides are displayed from phage and screened byaffinity chromatography to an immobilized receptor; see, e.g., WO91/17271, WO 92/01047, U.S. Pat. No. 5,223,409. In another approach,combinatorial libraries of polymers immobilized on a chip aresynthesized using photolithography; see, e.g., U.S. Pat. No. 5,143,854,WO 90/15070 and WO 92/10092. The immobilized polymers are contacted witha labeled receptor and scanned for label to identify polymers binding tothe receptor. The synthesis and screening of peptide libraries oncontinuous cellulose membrane supports that can be used for identifyingbinding ligands of the Futrin1, 2, 3 and/or -4 polypeptides and, thus,possible inhibitors and activators is described, for example, in Kramer,Methods Mol. Biol. 87 (1998), 25-39. This method can also be used, forexample, for determining the binding sites and the recognition motifs inthe Futrin 1, 2, 3 and/or -4 polypeptide. In like manner, the substratespecificity of the DnaK chaperon was determined and the contact sitesbetween human interleukin-6 and its receptor; see Rudiger, EMBO J. 16(1997), 1501-1507 and Weiergraber, FEBS Lett. 379 (1996), 122-126,respectively. Furthermore, the above-mentioned methods can be used forthe construction of binding supertopes derived from the Futrin 1, 2, 3or 4 polypeptide. A similar approach was successfully described forpeptide antigens of the anti-p24 (HIV-1) monoclonal antibody; seeKramer, Cell 91 (1997), 799-809. A general route to fingerprint analysesof peptide-antibody interactions using the clustered amino acid peptidelibrary was described in Kramer, Mol. Immunol. 32 (1995), 459-465. Inaddition, antagonists of a Futrin 1, 2, 3 and/or 4 polypeptide can bederived and identified from monoclonal antibodies that specificallyreact with a Futrin 1, 2, 3 and/or 4 polypeptide in accordance with themethods as described in Doring, Mol. Immunol. 31 (1994), 1059-1067.

All these methods can be used in accordance with the present inventionto identify activators/agonists and inhibitors/antagonists of a Futrin1, 2, 3 and/or 4 polypeptide.

Various sources for the basic structure of such an activator orinhibitor can be employed and comprise, for example, mimetic analogs ofa Futrin 1, 2, 3 and/or 4 polypeptide. Mimetic analogs of a Futrin 1, 2,3 and/or 4 polypeptide or biologically active fragments thereof can begenerated by, for example, substituting the amino acids that areexpected to be essential for the biological activity with, e.g.,stereoisomers, i.e. D-amino acids; see e.g., Tsukida, J. Med. Chem. 40(1997), 3534-3541. Furthermore, in case fragments are used for thedesign of biologically active analogs pro-mimetic components can beincorporated into a peptide to reestablish at least some of theconformational properties that may have been lost upon removal of partof the original polypeptide; see, e.g., Nachman, Regul. Pept. 57 (1995),359-370. Furthermore, a Futrin 1, 2, 3 and/or 4 polypeptide can be usedto identify synthetic chemical peptide mimetics that bind to or canfunction as a ligand, substrate or binding partner of saidpolypeptide(s) as effectively as does the natural polypeptide; see,e.g., Engleman, J. Clin. Invest. 99 (1997), 2284-2292. For example,folding simulations and computer redesign of structural motifs of aFutrin 1, 2, 3 and/or 4 polypeptide can be performed using appropriatecomputer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman,Comput. Appl. Biosci. 11 (1995), 675-679). Computer modeling of proteinfolding can be used for the conformational and energetic analysis ofdetailed peptide and protein models (Monge, J. Mol. Biol. 247 (1995),995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). Inparticular, the appropriate programs can be used for the identificationof interactive sites of a Futrin 1, 2, 3 and/or 4 polypeptide and itsligand or other interacting proteins by computer assistant searches forcomplementary peptide sequences (Fassina, Immunomethods 5 (1994),114-120. Further appropriate computer systems for the design of proteinand peptides are described in the prior art, for example in Berry,Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci.501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The resultsobtained from the above-described computer analysis can be used for,e.g., the preparation of peptide mimetics of a Futrin 1, 2, 3 and/or 4polypeptide or fragments thereof. Such pseudopeptide analogues of thenatural amino acid sequence of the protein may very efficiently mimicthe parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224).For example, incorporation of easily available achiral ω-amino acidresidues into a Futrin 1, 2, 3 or -4 polypeptide or a fragment thereofresults in the substitution of amide bonds by polymethylene units of analiphatic chain, thereby providing a convenient strategy forconstructing a peptide mimetic (Banerjee, Biopolymers 39 (1996),769-777). Superactive peptidomimetic analogues of small peptide hormonesin other systems are described in the prior art (Zhang, Biochem.Biophys. Res. Commun. 224 (1996), 327-331). Appropriate peptide mimeticsof a Futrin 1, 2, 3 and/or 4 polypeptide can also be identified by thesynthesis of peptide mimetic combinatorial libraries through successiveamide alkylation and testing the resulting compounds, e.g., for theirbinding and immunological properties. Methods for the generation and useof peptidomimetic combinatorial libraries are described in the priorart, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, athree-dimensional and/or crystallographic structure of a Futrin 1, 2, 3and/or 4 polypeptide can be used for the design of peptide mimeticinhibitors of the biological activity of the polypeptide (Rose,Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4(1996), 1545-1558).

It is also well known to the person skilled in the art, that it ispossible to design, synthesize and evaluate mimetics of small organiccompounds that, for example, can act as a substrate or ligand to aFutrin 1, 2, 3 and/or 4 polypeptide. For example, it has been describedthat D-glucose mimetics of hapalosin exhibited similar efficiency ashapalosin in antagonizing multidrug resistance assistance-associatedprotein in cytotoxicity; see Dinh, J. Med. Chem. 41 (1998), 981-987.

The nucleic acid molecule encoding a Futrin 1, 2, 3 and/or 4 polypeptidecan also serve as a target for activators and inhibitors. Activators maycomprise, for example, proteins that bind to the mRNA of a gene encodinga Futrin 1, 2, 3 and/or 4 polypeptide, thereby stabilizing the nativeconformation of the mRNA and facilitating transcription and/ortranslation, e.g., in like manner as Tat protein acts on HIV-RNA.Furthermore, methods are described in the literature for identifyingnucleic acid molecules such as an RNA fragment that mimics the structureof a defined or undefined target RNA molecule to which a compound bindsinside of a cell resulting in retardation of cell growth or cell death;see, e.g., WO 98/18947 and references cited therein. These nucleic acidmolecules can be used for identifying unknown compounds ofpharmaceutical interest, and for identifying unknown RNA targets for usein treating a disease. These methods and compositions can be used inscreening for novel or for identifying compounds useful to alterexpression levels of polypeptides encoded by a nucleic acid molecule.Alternatively, for example, the conformational structure of the RNAfragment which mimics the binding site can be employed in rational drugdesign to modify known drugs to make them bind more avidly to thetarget. One such methodology is nuclear magnetic resonance (NMR), whichis useful to identify drug and RNA conformational structures. Stillother methods are, for example, the drug design methods as described inWO 95/35367, U.S. Pat. No. 5,322,933, where the crystal structure of theRNA fragment can be deduced and computer programs are utilized to designnovel binding compounds.

The compounds which can be tested and identified according to a methodof the invention may be expression libraries, e.g., cDNA expressionlibraries, peptides, proteins, nucleic acids, antibodies, small organiccompounds, hormones, peptidomimetics, PNAs or the like (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198 and references cited supra). Furthermore, genesencoding a putative regulator of a Futrin 1, 2, 3 and/or 4 polypeptideand/or which exert their effects up- or downstream a Futrin 1, 2, 3and/or 4 polypeptide may be identified using, for example, insertionmutagenesis using, for example, gene targeting vectors known in the art.Said compounds can also be functional derivatives or analogues of knowninhibitors or activators. Such useful compounds can be for exampletransacting factors which bind to a Futrin 1, 2, 3 and/or 4 polypeptideor regulatory sequences of the gene encoding it. Identification oftransacting factors can be carried out using standard methods in the art(see, e.g., Sambrook, supra). To determine whether a protein binds tothe protein itself or regulatory sequences, standard native gel-shiftanalyses can be carried out. In order to identify a transacting factorwhich binds to the protein or regulatory sequence, the protein orregulatory sequence can be used as an affinity reagent in standardprotein purification methods, or as a probe for screening an expressionlibrary. The identification of nucleic acid molecules which encodepolypeptides which interact with a Futrin 1, 2, 3 and/or 4 polypeptidedescribed above can also be achieved, for example, as described inScofield (Science 274 (1996), 2063-2065) by use of the so-called yeast“two-hybrid system”. In this system the Futrin1, 2, 3 or 4 polypeptideor a smaller part thereof is linked to the DNA-binding domain of theGAL4 transcription factor. A yeast strain expressing this fusionpolypeptide and comprising a lacZ reporter gene driven by an appropriatepromoter, which is recognized by the GAL4 transcription factor, istransformed with a library of cDNAs which will express plant proteins orpeptides thereof fused to an activation domain. Thus, if a peptideencoded by one of the cDNAs is able to interact with the fusion peptidecomprising a peptide of a Futrin 1, 2, 3 and/or 4 polypeptide, thecomplex is able to direct expression of the reporter gene. In this waythe nucleic acid molecules encoding Futrin1, 2, 3 and 4, respectively,and the encoded peptide can be used to identify peptides and proteinsinteracting with a Futrin1, 2, 3 and/or 4 polypeptide.

Once the transacting factor is identified, modulation of its binding toor regulation of expression of a Futrin 1, 2, 3 and/or 4 polypeptide canbe pursued, beginning with, for example, screening for inhibitorsagainst the binding of the transacting factor to a Futrin1, 2, 3 or 4polypeptide. Activation or repression of a Futrin 1, 2, 3 and/or 4polypeptide could then be achieved in animals by applying thetransacting factor (or its inhibitor) or the gene encoding it, e.g. inan expression vector. In addition, if the active form of the transactingfactor is a dimer, dominant-negative mutants of the transacting factorcould be made in order to inhibit its activity. Furthermore, uponidentification of the transacting factor, further components in thesignal cascade leading to activation (e.g. signal transduction) orrepression of a gene involved in the control of a Futrin 1, 2, 3 and/or4 polypeptide then can be identified. Modulation of the activities ofthese components can then be pursued, in order to develop additionaldrugs and methods for modulating the metabolism of protein degradationin animals. Thus, the present invention also relates to the use of thetwo-hybrid system as defined above for the identification of activatorsor inhibitors of a Futrin 1, 2, 3 and/or 4 polypeptide.

The compounds isolated by the above methods also serve as lead compoundsfor the development of analog compounds. The analogs should have astabilized electronic configuration and molecular conformation thatallows key functional groups to be presented to a Futrin 1, 2, 3 and/or4 polypeptide or its ligand in substantially the same way as the leadcompound. In particular, the analog compounds have spatial electronicproperties which are comparable to the binding region, but can besmaller molecules than the lead compound, frequently having a molecularweight below about 2 kD and preferably below about 1 kD. Identificationof analog compounds can be performed through use of techniques such asself-consistent field (SCF) analysis, configuration interaction (CI)analysis, and normal mode dynamics analysis. Computer programs forimplementing these techniques are available; e.g., Rein,Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss,New York, 1989). Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, New York, USA. Furthermore, said derivativesand analogues can be tested for their effects according to methods knownin the art; see also supra. Furthermore, peptidomimetics and/or computeraided design of appropriate derivatives and analogues can be used, forexample, according to the methods described above.

Once the described compound has been identified and obtained, it ispreferably provided in a therapeutically acceptable form.

Accordingly, the present invention also relates to a pharmaceuticalcomposition comprising a nucleic acid molecule encoding a Futrin 1, 2, 3and/or 4 polypeptide, a Futrin 1, 2, 3 and/or 4 polypeptide itself,recombinant vector (for examples, see below), antibody,activator/agonist, inhibitor/antagonist and/or binding partner of aFutrin 1, 2, 3 and/or 4 polypeptide and a pharmaceutically acceptableexcipient, diluent or carrier.

Preferably, for therapeutic purposes, the Futrin 1, 2, 3 and/or 4polypeptide is recombinantly produced by use of the nucleic acidsequences shown in FIGS. 1 and 2. Suitable vectors for recombinantexpression are known to the person skilled in the art. Preferably, theyare plasmids, cosmids, viruses, bacteriophages and other vectors usuallyused in the field of genetic engineering. Vectors suitable for use inthe present invention include, but are not limited to the T7-basedexpression vector for expression in mammalian cells andbaculovirus-derived vectors for expression in insect cells. Preferably,the nucleic acid molecule of the invention is operatively linked to theregulatory elements in the recombinant vector of the invention thatguarantee the transcription and synthesis of an mRNA in prokaryoticand/or eukaryotic cells that can be translated. The nucleotide sequenceto be transcribed can be operably linked to a promoter like a T7,metallothionein I or polyhedrin promoter. The host cells used forrecombinant expression are prokaryotic or eukaryotic cells, for examplemammalian cells, bacterial cells, insect cells or yeast cells. Thepolypeptide is isolated from the cultivated cells and/or the culturemedium. Isolation and purification of the recombinantly producedpolypeptide may be carried out by conventional means includingpreparative chromatography and affinity and immunological separationsusing, e.g., an anti-Futrin 1, 2, 3 or -4 antibody, or, e.g., can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67; 31-40 (1988).

Examples of suitable pharmaceutical carriers etc. are well known in theart and include phosphate buffered saline solutions, water, emulsions,such as oil/water emulsions, various types of wetting agents, sterilesolutions etc. Such carriers can be formulated by conventional methodsand can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular,topical or intradermal administration. The route of administration, ofcourse, depends on the nature of the disease and the kind of compoundcontained in the pharmaceutical composition. The dosage regimen will bedetermined by the attending physician and other clinical factors. As iswell known in the medical arts, dosages for any one patient depends onmany factors, including the patient's size, body surface area, age, sex,the particular compound to be administered, time and route ofadministration, the kind and stage of the disease, e.g., tumor, generalhealth and other drugs being administered concurrently.

The delivery of the nucleic acid molecules encoding a Futrin 1, 2, 3and/or 4 polypeptide can be achieved by direct application or,preferably, by using a recombinant expression vector such as a chimericvirus containing these compounds or a colloidal dispersion system.Direct application to the target site can be performed, e.g., byballistic delivery, as a colloidal dispersion system or by catheter to asite in artery. The colloidal dispersion systems which can be used fordelivery of the above nucleic acid molecules include macromoleculecomplexes, nanocapsules, microspheres, beads and lipid-based systemsincluding oil-in-water emulsions (mixed), micelles, liposomes andlipoplexes, The preferred colloidal system is a liposome. Organ-specificor cell-specific liposomes can be used in order to achieve delivery onlyto the desired tissue. The targeting of liposomes can be carried out bythe person skilled in the art by applying commonly known methods. Thistargeting includes passive targeting (utilizing the natural tendency ofthe liposomes to distribute to cells of the RES in organs which containsinusoidal capillaries) or active targeting (for example by coupling theliposome to a specific ligand, e.g., an antibody, a receptor, sugar,glycolipid, protein etc., by well known methods). In the presentinvention monoclonal antibodies are preferably used to target liposomesto specific tissues, e.g. tumor tissue, via specific cell-surfaceligands.

Preferred recombinant vectors useful for gene therapy are viral vectors,e.g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNAvirus such as a retrovirus. Even more preferably, the retroviral vectoris a derivative of a murine or avian retrovirus. Examples of suchretroviral vectors which can be used in the present invention are:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV) and Rous sarcoma virus(RSV). Most preferably, a non-human primate retroviral vector isemployed, such as the gibbon ape leukemia virus (GaLV), providing abroader host range compared to murine vectors. Since recombinantretroviruses are defective, assistance is required in order to produceinfectious particles. Such assistance can be provided, e.g., by usinghelper cell lines that contain plasmids encoding all of the structuralgenes of the retrovirus under the control of regulatory sequences withinthe LTR. Suitable helper cell lines are well known to those skilled inthe art. Said vectors can additionally contain a gene encoding aselectable marker so that the transduced cells can be identified.Moreover, the retroviral vectors can be modified in such a way that theybecome target specific. This can be achieved, e.g., by inserting apolynucleotide encoding a sugar, a glycolipid, or a protein, preferablyan antibody. Those skilled in the art know additional methods forgenerating target specific vectors. Further suitable vectors and methodsfor in vitro- or in vivo-gene therapy are described in the literatureand are known to the persons skilled in the art; see, e.g., WO 94/29469or WO 97/00957.

In order to achieve expression only in the tar-get organ, e.g., a tumorto be treated, the nucleic acid molecules encoding a Futrin 1, 2, 3and/or 4 polypeptide can be linked to a tissue specific promoter andused for gene therapy. Such promoters are well known to those skilled inthe art (see e.g. Zimmermann et al., (1994) Neuron 12, 11-24; Vidal etal.; (1990) EMBO J. 9, 833-840; Mayford et al., (1995), Cell 81,891-904; Pinkert et al., (1987) Genes & Dev. 1, 268-76).

The present invention also relates to the use of the above compounds ofthe invention for the preparation of a pharmaceutical composition fortreatment of a disease associated with (a) aberrant expression of Futrin1, 2, 3 and/or 4 and/or genes involved into the Wnt signal cascade,and/or (b) aberrant activities or amounts of a Futrin 1, 2, 3 and/or 4and/or a polypeptide involved into the Wnt signal cascade. In apreferred embodiment, said disease is a kidney, bone or muscle diseaseor tumor, preferably breast cancer, a colon carcinoma or a melanoma.

Finally, the present invention relates to the use of a nucleotidemolecule encoding a polypeptide having a biological activity of Futrin1, 2, 3 and/or 4, a Futrin 1, 2, 3 and/or 4 polypeptide, anactivator/agonist of a Futrin 1, 2, 3 and/or 4 polypeptide or bindingpartner of said polypeptide (s) for the preparation of a pharmaceuticalcomposition for inhibiting the Wnt signal cascade which might be usefulfor supporting regenerative processes in a patient, e.g. growth oftissue like muscle, bone, hair, etc.

The following examples illustrate the invention.

Example 1 Materials and Methods

(A) Isolation of Futrins (=R-spondins) and Constructs

A Xenopus adult eye cDNA library in pCS2+ was used to prepare pools ofabout 250 colonies. Plasmid DNA from each pool was transientlytransfected into 293T cells together with the Wnt receptor frizzled8,the Wnt reporter TOPFLASH (Korinek et al., Science 275 (1997), 1784-7)and pRL-TK (Promega) using FuGENE6 (Roche) transfection reagent.Luciferase assay was carried out 24 hours after transfection. A positiveclone was isolated from the pool by sib selection. Human Rspo2 and 3cDNAs were obtained from RZPD. Fragments of hRspo1 and 4 were RT-PCRamplified from mRNA of 293T cells and used as hybridization probes. Fulllength mouse Rspo1 and 2 were isolated from a mouse embryonic day 13.5cDNA library. The sequence of X. tropicalis Rspo3 was obtained fromSanger Institute database and a cDNA fragment was cloned by RT-PCR fromX. tropicalis embryos. C-terminally Myc or FLAGtagged constructs and alldeletion constructs were created by PCR. Xenopus Rspo2ΔC was cloned bydeleting the last 37 amino acids. The Rspo3 cDNAs were cloned in pCS2+and Bluescript vectors for use in gene expression and as probes,respectively.

(B) Cell Culture, Recombinant Proteins and Luciferase Reporter Assays

HEK293T, SHEP and HeLa cell lines were maintained in DMEM, 10% FCS and10% CO₂ . Xenopus Rspo2ΔC conditioned medium was produced by transienttransfection in 293T cells. Mouse Wnt3a conditioned medium was producedfrom mouse L cells stably transfected with Wnt3a (ATCC#CRL-2647)(Shibamoto et al., Genes Cells 3 (1998), 659-670). Luciferase reporterassays in 293T cells were carried out in 96-well plates as described (Wuet al., Curr. Biol. 10 (2000), 1611-1614). Luciferase reporter assays inHeLa cells were carried out in 24 well plates in triplicates usingLipofectamine Plus transfection reagent (Invitrogen). Per well a totalof 400 ng DNA were transfected, including 80 ng 7lef-fos-Luc (Novak etal., PNAS 95 (1998), 4374-4379), 10 ng pRL-TK, 10 ng mouse frizzled8, 2ng mouse lef1 and 300 ng pSuper plasmid DNAs. 3 days after transfection,either mouse Wnt3a conditioned medium or medium containing 30 mM LICIwas added to stimulate Wnt signalling. 24 hours later, luciferaseactivity was determined using the Dual luciferase system (Promega).

(C) Embryos, Explants, In Situ Hybridization and RNA Synthesis

In vitro fertilisation, embryo culture, staging, microinjection andculture of Xenopus embryo explants were carried out as described(Gawantka et al., EMBO J. 14 (1995), 6268-79). Double- andsingle-labeling whole-mount in situ hybridization was carried outaccording to (Bradley et al., Development 122 (1996), 2739-50). A PCRfragment of tropicalis Rspo3 cDNA was used for in situ hybridization onXenopus laevis embryos. For vibratome sectioning, embryos were placed inembedding medium (0.4% gelatine, 30% albumin, 20% sucrose in PBS) andmounted in the presence of 2% glutaraldehyde. Sectioning was carried outusing a VT100E vibratome (Leica). Brains of 4 day Xenopus embryos wereexcised in 1× Barth solution and fixed for in situ hybridisation.Whole-mount in situ hybridisation of mouse embryos was performedaccording to previously described procedures (Koop et al., Mech. Dev. 59(1996), 73-78). Preparation of mRNA for Xenopus injections was carriedout using the MegaScript in vitro transcription kit (Ambion), accordingto the manufacturer's instructions.

(D) Morpholino Antisense Oligonucleotides and siRNA Constructs

The 5′ nucleotide sequence of an additional (pseudo-) allele for XenopusRspo2 gene was obtained using 5′ RACE (GeneRacer kit, Invitrogen). Basedon these sequences, an antisense morpholino oligonucleotide targetingboth pseudoalleles around the ATG start codon was designed (Rspo2Mo):GCCGTCCAAATGCAGTTTCAAC (SEQ ID NO:1). pSuper constructs producing siRNAagainst human Rspo 2, 3 or a non-sense control were made according toBrummelkamp et al., Science 296 (2002), 550-3. The sequences are: humanRspo2, TCCCATTTGCAAGGGTTGT (SEQ ID NO:2); human Rspo3,AGCTGACTGTGATACCTGT (SEQ ID NO:3); nonsense control, ACTACCGTTGTTATAGGTG(SEQ ID NO:4).

(E) Immunohistochemistry, Western Blot and Dot Blot Analysis

Immunohistochemistry to detect β-catenin in SHEP cells was carried outaccording to (Scheiffele et al., J. Cell. Biol. 140 (1998), 795-806)using anti-β-catenin antibody (Transduction laboratories, Newington).For detection of tagged Rspo proteins or loading controls on Westernblot, anti-Myc (clone 9E10), anti-FLAG (M2, SIGMA) monoclonalantibodies, chick anti-GFP (Chemicon, Hampshire) and mouseanti-α-tubulin (SIGMA) antibodies were used. Chemiluminescence detection(SuperSignal® solution, Pierce) was carried out according to themanufacturer's instructions after incubation of blots with anti-mouseIgG-HRP (Pierce). For Rspo expression analysis in tumour samples theCancer Profiling Array II (Clontech, Palo Alto) was used andhybridization was carried out according the manufacturer's instructions.

(F) RT-PCR

RT-PCR assays were carried out as described (Dosch et al., Development124 (1997), 2325-34; Glinka et al., Nature 389 (1997), 517-519);additional primers were: Xenopus Rspo2 (forward, GAATGCCCAGAAGGATTTGC(SEQ ID NO:5); reverse, GGGATGGTGTCTTTTGCTGG (SEQ ID NO:6)); XenopusRspo3 (forward, GAAGCAAATTGGAGTCTGTCG (SEQ ID NO:7); reverse,GATTGTTCTCAAACCCTTCAGG (SEQ ID NO:8)); human Rspo1 (forward,ACAGACACAAGACACACACGC (SEQ ID NO:9); reverse, TGTCTTCTGGTGGCCTCAG (SEQID NO:10)); human Rspo2 (forward, CCGAGCCCCAGATATGAAC (SEQ ID NO:11);reverse, TGACCAACTTCACATCCTTCC (SEQ ID NO:12)); human Rspo3 (forward,AGGGACTGAAACACGGGTC (SEQ ID NO:13); reverse, TGTCTTCTGGTGGCCTCAG (SEQ IDNO:14)); human Rspo4 (forward, AAGCTGGGACACAGCACAG (SEQ ID NO:15);reverse, GAAGCCTTGGAGCCTTGTC (SEQ ID NO:16)).

Example 2 Isolation of a cDNA Encoding Xenopus Futrin 1

A Xenopus adult eye cDNA library in the expression vector pCS2+ was usedto prepare pools of about 250 colonies, and plasmid DNA from each poolwas transiently transfected into 293T cells together with the Wntreceptor frizzled8, the Wnt reporter TOP-FLASH (Korinek et al. Science275 (1997) 1784-1787) and Renilla-luciferase for normalization, in96-well plates using FuGENE 6 (Roche, Basel). After 24 hours relativeluciferase activity was determined. One pool yielding a signal abovebackground was identified (FIGS. 1 and 2A) and a gene harboring thisactivity was isolated from the pool by sib selection. Sequencinganalysis showed it represents Xenopus futrin 1. Database searchesrevealed four closely related genes in human, called hfutrin 1, 2, 3 and4 (FIGS. 3A-D and 4 or 6A).

Xenopus futrin 1 (Rspo2) is predicted to encode a secreted protein with243 amino acids (mature protein) and an isoelectric point of 9.8. AllR-spondins contain an N-terminal signal peptide (SP), two furin-likedomains (FU), one thrombospondin type1 domain (TSP1) and a C-terminallow complexity region enriched with positively charged amino acids (C)(FIG. 2F). The furin-like cysteine rich domain is found in e.g.furin-like endoproteases, EGF receptor and insulin receptor. In signaltransduction by certain receptor tyrosine kinases furin repeats arerequired for receptor aggregation. TSP1 repeats are found inThrombospondin and other extracellular matrix proteins like Mindin,F-spondin, SCO-spondin as well as in a number of proteins involved inthe complement cascade. Proteins containing TSP1 repeats are involved incell-cell interaction, inhibition of angiogenesis and apoptosis (Adamsand Tucker, Dev. Dyn. 218 (2000), 280-99).

While Xenopus Rspo2 contains a predicted N-terminal signal peptide,secreted protein is almost undetectable in the medium of transientlytransfected 293T cells. Since the C-terminus is enriched with basicamino acids, which promotes cell surface retention a C-terminallytruncated protein was tested. Rspo2ΔC is effectively secreted into themedium from 293T cells (FIG. 2B) and is functionally active (FIG. 2F).Since all R-spondins share the basic C-terminus and since there are noobvious ER- or Golgi retention signals, this suggests that the proteinsare normally associated with the cell surface.

Example 3 Futrins Promote Wnt Signalling

Xenopus Futrin1 and human Futrin1, 2, and 3 are able to stimulateWnt-responsive reporter expression in HEK 293T cells when provided bytransient transfection (FIGS. 2C and 5A). In addition, they are able toenhance reporter expression induced by Wnt (mouse Wnt1/3A)synergistically (FIGS. 2D and 5B). Cotransfection experiments in HEK239Tcells were carried out with the indicated genes and the Wnt reporterTOP-FLASH (Korinek et al. Science 275 (1997) 1784-1787) andRenilla-luciferase for normalization, in 96-well plates using FuGENE 6(Roche). After 24 hours relative luciferase activity was determined.

All tested members of the Rspo family (e.g. murine Rspo1-3, human Rspo2,3) show equivalent effects (FIG. 6A and data not shown). Rspo2signalling is sensitive to the Wnt/β-catenin pathway inhibitors dominantnegative TCF and dickkopf1 (FIG. 6B). A synergistic signalling effect isobserved when Rspo2 is cotransfected with extracellular but not withintracellular components of the Wnt/β-catenin pathway (FIG. 6C). Thegreatest cooperation is reproducibly seen between Rspo2 and Wnts, eitherusing conditioned media (FIG. 2D) or following co-transfection (FIG.6C).

A hallmark of Wnt/β-catenin signalling activation is the cytosolicaccumulation of β-catenin due to its stabilisation. Treatment of 293Tcells with Wnt3a conditioned medium induces cytosolic β-catenin after 1hour and while recombinant Rspo2ΔC alone is not able to stabilizeβ-catenin during this interval, it strongly enhances activity of Wnt3ato do so (FIG. 2E, top). After 4 hours treatment, both Wnt3a and Rspo2ΔCconditioned media are able to induce β-catenin accumulation to similarlevels (FIG. 2E, top). β-catenin is known to enter the nuclei inresponse to Wnt stimulation and activate gene expression together withLef/Tcf transcription factors. Treatment of SHEP cells by either Wnt3aor Rspo2ΔC conditioned media weakly induces β-catenin nuclearlocalization, while co-treatment with Wnt3a and Rspo2ΔC stronglyenhances nuclear accumulation (FIG. 2E, bottom). It can be concludedthat R-spondins represent a novel family of secreted proteins capablepromoting Wnt/β-catenin signalling.

To functionally study its signalling domains, serial deletions ofXenopus Rspo2 (FIG. 2F) were created. As discussed, the basic C-terminuscan be removed without loss of activity and this is also true for theTSP1 domain (FIG. 2F). However, the furin-like domains are necessary forWnt/β-catenin signalling since deletion of either the furin-1 or -2domains abolishes the activity in reporter assays (FIG. 2F).

Example 4 Futrins are Required for Full Wnt Signalling

To test the requirement of Futrins in Wnt signalling, siRNA mediatedgene knock-out was utilized (Brummelkamp et al., Science. 2002, 296(5567): 550-3). Hela cells were transfected using Lipofectamine Pluswith 80 ng Wnt reporter 7LEF-Rev-fosLuc, 10 ng pRL-TK (Promega) and 300ng pSuper constructs (Brummelkamp et al.) that produce either siRNAagainst human Futrin1 and 2, or a nonsense control. 7LEF-Rev-fosLucreporter construct containing seven LEF binding sites in front ofminimal fos promoter followed by firefly luciferase ORF was kindlyprovided by R. Grosschedl (Howard Hughes Medical Institute). pSuperconstructs contain 19-nucleotide sequences from human Futrin1 (sequence:TCCCATTTGCAAGGGTTGT (SEQ ID NO:17)), human Futrin1 (sequence:AGCTGACTGTGATACCTGT (SEQ ID NO:18)) or control nonsense sequence(ACTACCGTTGTTATAGGTG (SEQ ID NO:19)).

One day after transfection medium was changed from 10% to 0.5% FCS.Three days after transfection, mouse Wnt3A conditioned medium or controlmedium from 293 cells was added to the culture to stimulate Wntsignalling. 24 hours later, luciferase activity was determined. As shownin FIG. 7, Hela cells show reduced levels of Futrin 1 and 2, and Wntsignalling dropped by 50%, indicating that Futrins are required for fullWnt signalling. This effect can be efficiently rescued by 5 ngrecombinant mouse Futrin 1, attesting its specificity. Data arenormalized to Renilla luciferase activity.

Example 5 Expression of R-Spondin Genes in Xenopus and Mouse Embryos

In Xenopus embryos, no maternal Rspo2 RNA is detected by RT-PCR. Itszygotic expression starts at early gastrula stage and remains constantthroughout neurulation and organogenesis (FIG. 8A). By whole-mount insitu hybridization, weak expression of Rspo2 is observed throughout theectoderm of early gastrulae (not shown). During gastrulation strongexpression is detected in the marginal zone in both deep and superficiallayers, but is excluded from the Spemann organizer (FIG. 8B). At lategastrula stage Rspo2 expression persists in lateral plate mesoderm andbecomes detectable in trie anterior neural plate (FIG. 8C). At stage 15expression is seen in two longitudinal stripes along the neural plate(future roof plate), in the anterior neural plate and in lateral andposterior mesoderm (FIG. 8D). Expression of Rspo2 at tailbud stage (FIG.8F) is restricted to several regions of the brain, includingdiencephalon and midbrain-hindbrain boundary, pronephros and dorsalneural tube. Expression is also detected in the dorsal and ventral-mostportions of somites, the dorsal fin and the proctodeum. Rspo2 expressionin the brain of late tadpoles is mainly restricted to diencephalon,including the zona limitans intrathalamica (zli) (FIG. 8H).

Xenopus Rspo3 expression is related to that of Rspo2. It is firstdetected at gastrula stage (not shown) and in neurulae it is expressedin the anterior border of the neural plate and posterior mesoderm (FIG.8E). At tail bud stage, it is coexpressed with Rspo2 in the centralnervous system but shows additional expression in branchial arches andthe tailbud (FIG. 8G).

In mouse, Rspo3 transcripts are detected by in situ hybridization at day7.5 in the primitive streak (FIG. 8J) while expression of Rspo1 and 2 isnot detectable. At day 9.5, Rspo1-3 show differential expression invarious neural and mesodermal derivatives (FIG. 8K-M), mainly alongdorsal neural tube (Rspo1 and 3), diencephalon (Rspo1, 2, 3), somites(Rspo3) and tailbud mesoderm (Rspo3). In limb buds all three genes showprominent differential expression (FIG. 8I), particularly, in themorphogenetically active region such as the apical ectodermal ridge(AER) (Rspo2).

Example 6 R-Spondins are Co-Expressed with and Regulated by Wnts

R-spondins not only show functional interaction with Wnt signalling, butalso co-expression with Wnt genes in many regions during Xenopus andmouse embryonic development. In gastrula mesoderm of both Xenopus andmouse, Rspo2 and -3 are co-expressed with XWnt8 and mWnt3, respectively.Similarly, at later stages, R-spondin family members are widelyco-expressed with a number of Wnt genes e.g. in midbrain-hindbrainboundary, dorsal neural tube and limb bud and tail bud. A directcomparison between the expression patterns of Xenopus Rspo2 and Wnt8 andWnt3a shows a large overlap (FIG. 9A).

Indeed, Wnts are able to induce Rspo expression since Xenopus embryosinjected with pCS-Wnt8 or pCS-β-catenin DNA upregulate both Rspo2 andRspo3 by RT-PCR (FIG. 9B).

Likewise, embryos injected with pCS-Wnt8 or pCS-Wnt3a DNA show ectopicRspo2 expression by in situ hybridization (FIG. 8C-E). The resultsindicate that the observed co-expression is due to regulation ofR-spondins by Wnts. This is consistent with the observation that Rspo1expression is reduced in dorsal spinal cord of Wnt1 or Wnt3a knockoutmouse embryos.

Example 7 Futrin 1 (R-Spondin 2) Activates Neural Markers and Regulates(Promotes) Muscle Formation

Loss-of-function analysis in Xenopus embryos show that Futrin 1 isrequired for muscle formation. Injections of antisence morpholino oligoagainst futrin 1 (Futl-Mo) cause downregulation of early muscle markersMyoD and Myf5 and induce muscle defects (FIG. 10A). The specificity ofthis effect was documented, first, by the ability of Fut1-Mo to inhibittranslation of the cognate DNA construct when overexpressed in embryos(FIG. 10B) and, second, by the ability of XFut1mRNA to rescue the effectof Fut1-Mo (FIG. 10C).

When the Wnt/(β-catenin pathway is overactivated in Xenopus embryos avariety of responses are observed: i) mRNA injection of pathwayactivators typically induces secondary embryonic axes in whole embryosand anterior neural markers in animal caps and whole embryos; ii) DNAinjection of pathway activators driving expression after MBT,posteriorizes the central nervous system (CNS). To test if Rspo2 is ableto mimic any of these effects synthetic mRNA was microinjected inXenopus embryos. However, Rspo2 mRNA injection does not induce secondaryaxes in whole embryos and injection of pCS-Rspo2 DNA does notposteriorize CNS, since heads are normal sized, Otx2 expression isexpanded and en2 unaffected (FIG. 11D, H).

In animal caps Rspo2 induces the pan-neural markers NCAM and N-tubulinand the anterior neural marker, Otx2, as do Xwnt6 and β-catenin (FIG.11A). Similarly, in whole embryos injected with Rspo2 mRNA, ectopiccement glands and lateral expansion of the neural plate are observed atthe injected side, as shown by expression of neural markers Sox3, Otx2,not2, en2 and Rx1 (FIGS. 11B-H). Ectopic Otx2 expression is observedalready in gastrula stage embryos (FIG. 11I+J). These results areconsistent with the ability of Wnt/β-catenin signalling to block BMP4expression and thereby to activate neural development.

To further analyze the mRNA overexpression effect of Rspo2 an BMP4,Activin, Nodal and FGF signaling, animal cap assays were carried out andtested for the induction of target genes by three growth factor signals(FIG. 12). Rspo2 expectedly blocks BMP4 mediated Xvent2 expression butsurprisingly also inhibits Activin and Nodal mediated Xbra induction(FIG. 12A-C). This is unlike XWnt8 and β-catenin mRNA injections, whichdo not affect Xbra induction by Activin (FIG. 12B). In contrast tosignaling by three TGF-β type growth factors, FGF8 induced Xbraexpression is unaffected by overexpressed Rspo2 (FIG. 12D). It can beconcluded that Rspo2 overexpression in addition to activating theWnt/β-catenin pathway, is also able to interfere with signaling by threeTGF-β family growth factors.

Another well known effect of zygotic Wnt/β-catenin signalling is itsability to promote myogenesis. For example, XWnt8 can induce muscleformation in ventral mesodermal cells. When ventral marginal zones(VMZs) from Rspo2 injected embryos are dissected and cultured untilstage 40, they elongate, form tail like structures, and are contractile.This phenotype is indistinguishable from control lateral marginal zoneexplants (LMZs), which typically differentiate muscle (FIG. 4K-L). Insitu hybridization confirmed induction of muscle actin in both Wnt8 andRspo2 injected VMZs but not in control (preprolactin-injected) VMZs(FIG. 11M). Since R-spondins are able to enhance Wnt signalling in 293Tcells, their cooperation in Xenopus myogenesis was tested. Myf5 is amyogenic marker characteristically expressed in lateral mesoderm.However, it is excluded from dorsal mesoderm, and dorsal marginal zone(DMZ) explants express only little myf5 as determined by RT-PCR (FIG.11N). In DMZs from embryos injected with DNA constructs driving post MBTexpression, myf5 is weakly induced by Rspo2, moderately by Wnt8 andstrongly by their combination (FIG. 11N, top). The myogenesis promotingeffect of Rspo2 is repressed by dominant negative dishevelled (Xddl),dkkl and GSK-3β, which all block Wnt signalling (FIG. 11N, bottom). Insummary, the results suggest that Rspo2 can promote myogenesis via theWnt/β-catenin pathway.

Example 8 R-Spondin2 is Required for Wnt/β-Catenin-Mediated Myogenesis

To investigate the physiological role of Rspo2 during Xenopusembryogenesis, antisense morpholino oligonucleotides were injected(Rspo2Mo). The ability of Rspo2Mo to block Rspo2 protein production isdemonstrated by Western blot (FIG. 13A). Injection of this Morpholinointo one dorso-animal blastomere at eight cell stage results in eyedefects, although the expression of early eye-(Rx1, Pax6), anteriorneural (Otx2) and pan-neural markers (Sox3) is not obviously affected(not shown).

Equatorial Injection of Rspo2Mo in one blastomere at eight cell stageleads to muscle defects at the injected side (FIG. 13B). In situhybridisation for muscle actin and transverse trunk sections show thatRspo2Mo injection causes disorganized somites (panels a-b) and reducedmyotomes (panels c-d). Lineage tracing experiments showed that Rspo2Moinjected cells contribute to lateral plate mesoderm instead of somitesor undergo cell death (data not shown). At gastrula stage expression ofthe myogenic markers myf5 and myoD is strongly down-regulated at theRspo2Mo injected side (panels e-h), while the pan-mesodermal marker Xhraand the organizer marker Xnot2 are unaffected (panels i-i).

To test the specificity of Rspo2Mo, rescue experiments were performed byco-injecting Rspo2Mo together with a Rspo2 RNA, in which six non-codingnucleotides were mutated so that it would not be targeted by thisMorpholino. Expression of myf5 (FIG. 13C) and myoD (data not shown) iseffectively rescued by this mutant Rspo2 RNA, indicating that the effectof Rspo2Mo an myogenesis is specific.

Rspo2Mo was used as a tool to examine the epistatic position of Rspo inthe Wnt/(β-catenin pathway. As read out for Wnt/β-catenin signalling theexpression of myf5 and muscle actin in marginal zone explants was used(FIG. 13D). In DMZ and VMZ explants, Wnt8 DNA induces myf5 and muscleactin and this induction is significantly blocked by Rspo2Mo (FIG. 13D,panels a-b, lanes 3-4). However, myf5 and muscle actin induction byintracellular activators of the Wnt pathway like dishevelled, dnGSK-36and β-catenin is unaffected by Rspo2Mo (a-b, lanes 5-10). In LMZexplants, Rspo2Mo Injection down-regulates endogenous myf5 and muscleactin expression (FIG. 13D, panels c-d, lanes 1-2). In DNA coinjectionsthis effect is rescued effectively by β-catenin—but only poorly by XWnt8(lanes 3-6). The residual effect of XWnt8 is likely due to its non-cellautonomous action an cells that did not receive Rspo2Mo. Taken togetherthese results indicate that Rspo2 affects the Wnt/(β-catenin pathway atthe level or upstream of dishevelled.

Example 9 R-Spondins are Required for Wnt/β-Catenin Signalling in HeLaCells

Next the requirement of Rspo2 for Wnt signalling was tested in mammaliancells using siRNA. Since there are four R-spondins with apparentlyredundant function, HeLa cells were selected, that only express Rspo3and very weakly Rspo2 (FIG. 14A). Various other cell lines, such as 293Tcells, express all four genes, complicating a siRNA approach.

siRNA mediated gene knock-down was carried out by transfecting pSUPERconstructs (Brummelkamp et al., Science 296 (2002), 3286-3305) toproduce siRNAs targeted against Rspo2 and -3 (siRNA Rspo 2, 3). Ascontrol, a non-sense siRNA was used. To test the efficiency of siRNA,FLAGtagged human Rspo3 was co-transfected with siRNAs and its productionwas repressed by siRNA Rspo3 but not siRNA Rspo2 (FIG. 14B).

In Wnt-reporter assays, both siRNA Rspo2 and -3 decreased Wnt3a-inducedluciferase activity compared to control siRNA (FIG. 14C). When siRNARspo2 and -3 are co-transfected, reporter activity drops to 40%. Thiseffect is specific since the decreased Wnt signalling can be rescued byco-transfected mouse Rspo2, which is not targeted by siRNAs againsthuman R-spondins (FIG. 14C). Furthermore, unlike Wnt3a-induced reporteractivity, siRNA Rspo2+3 do not affect Li+-induced Wnt/(β-cateninsignalling (FIG. 14D). Since Li+ acts by inhibiting GSK3β activity, thisis again consistent with Rspo2 acting at the level or upstream ofdishevelled. conclude that in HeLa cells R-spondins are required forfull Wnt/(β-catenin signalling.

Example 10 Futrin Expression is Deregulated in Human Tumors

Misregulation of Wnt/β-catenin signalling is implicated intumorigenesis, e.g., colon cancer, breast cancer and melanoma (Barker etal., 2000; Bienz and Clevers, 2000; Polakis, 2000). Since R-spondins(futrins) promote Wnt/β-catenin signalling they may also play a role intumorigenesis. Thus, the expression of Futrin 1-4 in various normal andcancerous human tissues was studied using radioactive hybridisation onClontech Cancer Profiling Array II. Hybridization with ubiquitin probewas used for normalisation. The results show that expression of Futrins1-3 is dramatically deregulated in cancerous human tissues (FIG. 15). Inmost of the tumors the expression of Futrins 1-3 is dramaticallydecreased (colon, stomach, lung, rectum tumors for Futrin 1, breast,ovary, bladder, uterus, cervix, rectum tumors for Futrin 2, uterus andcervix tumors for Futrin 3). In a few cases the expression of Futrin 1-3is upregulated (one case of stomach tumor for Futrin 1 and 2, ovarytumor for Futrin 3). Futrin 4 shows very low level of expression in mostof the tissues studied, except ovary. Further results are shown in FIG.16 also showing that HRspo1 is weakly expressed in adult organs. Itshows upregulation in ovary tumours from two patients and in one stomachtumour sample. HRspo2 is expressed in organs of endodermal origin,including colon, rectum, small intestine and lung. Its expressiondecreased in corresponding tumour samples. HRspo3 is expressed widelyand decreases in many tumour samples. In general, the expression ofhRspo1-3 is deregulated in a number of tumours, while hRspo4 expressionis very weak both in human adult organs and tumours (data not shown).

The invention claimed is:
 1. A method of determining whether a bindingpartner of a Futrin 1 polypeptide affects Wnt signaling activity, themethod comprising: (a) providing a mammalian cell in culture comprisingthe Futrin 1 polypeptide and a Wnt inducible reporter, wherein theFutrin 1 polypeptide comprises the amino acid sequence of SEQ ID NO:26,the amino acid sequence of amino acids 21-243 of SEQ ID NO:26, the aminoacid sequence of amino acids 1-146 of SEQ ID NO:26, or the amino acidsequence of amino acids 21-146 of SEQ ID NO:26, wherein the Futrin 1polypeptide is able to promote Wnt signaling; and (b) detecting thelevel of reporter expression in the cell in the presence and absence ofthe binding partner, wherein a change in the level of reporterexpression indicates that the binding partner affects the Wnt signaling.2. The method of claim 1, wherein the Futrin 1 polypeptide comprises theamino acid sequence of amino acids 21-243 of SEQ ID NO:26.
 3. The methodof claim 1, wherein the Futrin 1 polypeptide comprises SEQ ID NO:26. 4.The method of claim 1, wherein the Futrin 1 polypeptide comprises theamino acid sequence of amino acids 1-146 of SEQ ID NO:26.
 5. The methodof claim 1, wherein the Futrin 1 polypeptide of comprises the amino acidsequence of amino acids 21-146 of SEQ ID NO:26.
 6. The method of claim1, wherein the change is a decrease in the level of reporter expressionand the decrease indicates that the binding partner is an antagonist ofWnt signaling.
 7. The method of claim 6, wherein the binding partner isan antibody.
 8. The method of claim 7, wherein the antibody is amonoclonal antibody.
 9. The method of claim 8, wherein the monoclonalantibody is a chimeric or a humanized antibody.
 10. The method of claim9, wherein the Futrin 1 polypeptide comprises the amino acid sequence ofamino acids 21-243 of SEQ ID NO:26.
 11. The method of claim 9, whereinthe Futrin 1 polypeptide comprises SEQ ID NO:26.
 12. The method of claim9, wherein the Futrin 1 polypeptide comprises the amino acid sequence ofamino acids 1-146 of SEQ ID NO:26.
 13. The method of claim 9, whereinthe Futrin 1 polypeptide comprises the amino acid sequence of aminoacids 21-146 of SEQ ID NO:26.
 14. The method of claim 1, wherein thechange is an increase in the level of reporter expression and theincrease indicates that the binding partner is an agonist of Wntsignaling.